Polypeptides containing polymorphisms of the repeated regions of pertactin in bordetella pertussis, bordetella parapertussis, and bordetella bronchiseptica, their use in diagnostics, and in immunogenic compositions

ABSTRACT

Pertactin (PRN) is an outer membrane protein expressed by  Bordetella pertussis, Bordetella parapertussis,  and  Bordetella bronchiseptica,  which induces protective immunity to Bordetella infections. The immunodominant and immunoprotective epitopes of pertactin include two repeated regions, I and II. Comparison of these two repeated regions showed the pertactin of  B. parapertussis  is invariant, whereas the pertactin of  B. pertussis  varies mostly in region I and  B. bronchiseptica  varies in both the repeated regions I and II. Compositions containing pertactins and pertactin fragments containing variant sequences in these regions are useful as immunogenic compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims the benefit of U.S.Provisional Application Ser. No. 60/206,969, filed May 25, 2000(attorney docket no. 03495.6047) The entire disclosure of thisapplication is relied upon and incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] This invention relates to proteins and polypeptides of theBordetella outer membrane protein called pertactin and thepolynucleotides that encode them. This invention also relates to the useof these proteins and polypeptides in immunogenic compositions,diagnostic methods, and diagnostic kits.

[0003] The genus Bordetella includes seven species. The most studiedspecies are B. pertussis, B. parapertussis, and B. bronchiseptica. B.pertussis is responsible for respiratory infections only in humans. B.parapertussis causes infections in humans and sheep, and B.bronchiseptica infects many animal species, including humans.

[0004] These pathogens produce an array of virulence factors, thesynthesis of which is regulated by the two-component, bvg AS (2, 21)system. These factors include toxins, such as pertussis toxin, which isthe only toxin specific to B. pertussis, tracheal cytotoxin, adenylatecyclase-hemolysin, and adhesins, such as filamentous hemagglutinin,fimbriae, and pertactin (PRN).

[0005] PRN is an outer membrane protein with an apparent molecularweight of 69 kDa in B. pertussis, 70 kDa in B. parapertussis, and 68 kDain B. bronchiseptica (5, 14, 15). The precursors of PRN are 91.5 kDa, 93kDa, and 92.5 kDa in size, respectively. In B. pertussis, PRN has beendemonstrated to be an agglutinogen (4), promoting attachment to certaineukaryotic cells via an Arg-Gly-Asp (RGD) motif (13).

[0006] Antibodies specific for the B. bronchiseptica PRN are detected athigh titer in immunized piglets, whereas few if any of these antibodiesare detected in unprotected animals (19). Synthesis of the PRN by B.bronchiseptica correlates with protection (16). The immunization of miceor piglets with preparations of the PRN induces protective immunityagainst B. bronchiseptica infection (12, 19) and passively administeredmonoclonal antibodies prevent the death of animals challenged with B.bronchiseptica (16). B. pertussis PRN has also been shown to induceprotective immunity to intracerebral, aerosol and intranasal challengeswith B. pertussis in mice (11, 18, 20).

[0007] PRN is, therefore, now included in some acellular pertussisvaccines (i.e. vaccines composed of purified bacterial proteins) (9).However, the PRN proteins of these three species, although clearlyrelated, have different immunogenic properties. For example,preparations of B. pertussis PRN protect mice against intranasal B.pertussis challenge but not against intranasal B. parapertussischallenge (11). They also protect mice against intracerebral B.pertussis challenge, whereas the B. bronchiseptica PRN protein does not(18).

[0008] Comparison of the deduced amino acids of the three proteins, B.pertussis-PRN, B. parapertussis-PRN, and B. bronchiseptica-PRN, revealsa high degree of similarity, with the B. bronchiseptica and B.parapertussis proteins being more similar to each other than to the B.pertussis PRN protein (5, 14, 15).

[0009] The sequences of the three proteins differ in the number ofrepeats in regions I and II (FIG. 1a). Using monoclonal antibodies,Charles et al., identified and characterized a protective immunodominantepitope of the P.69-PRN protein (6). This epitope spans the(Pro-Gln-Pro)5 repeat sequences located in region II. Differences inthis region may account for the observation that sera from piglets thatrecognize B. bronchiseptica PRN do not react with B. pertussis PRNdespite the high degree of similarity between these proteins (12) andfor the lack of cross protection provided by the three proteins (11, 18,20).

[0010] It has recently been shown that the PRN produced by clinicalisolates of B. pertussis varies. Sequences of the prn gene of variousclinical isolates revealed three major types of PRN variant (17). It hasbeen suggested that epidemics in the Netherlands result from changes inthe sequences of the genes encoding PRN and PT because the proteinspresent in the clinical isolates currently in circulation differ insequence from those observed by the vaccinal strains used in thiscountry (17).

[0011] For PRN of B. pertussis, all the observed amino acid differencesare located in region I. The allelic prn types A=1 and C=3 are verysimilar, differing by only two amino acids, whereas type B=2 is quitedifferent, having a five-amino acid insertion in the same region (17).

[0012] Only one type was found to differ in region II. This type (A*=6)is produced by the B. pertussis WHO reference strain 18323 and oneFrench clinical isolate (3). It does not, however, seem to be commonbecause it has been detected in only one clinical isolate (3). Theproduction by this B. pertussis strain of this unusual type of PRNreflects the many common properties shared with the B. parapertussis andB. bronchiseptica species. No differences were found in the phenotypeand behavior in the animal model of B. pertussis clinical isolates withdifferent PRN (3).

[0013] There is a need in the art for compositions containing proteinsand polypeptides of Bordetella pertactins that can be used inimmunogenic compositions to protect against Bordetella infection and totreat subjects infected with Bordetella. Ideally, the proteins,polypeptides, and the polynucleotides that encode them would also beuseful in diagnosing Bordetella infection and in kits for the diagnosisof such infection.

SUMMARY OF THE INVENTION

[0014] This invention aids in fulfilling these needs in the art. In oneembodiment, this invention provides an immunogenic compositioncomprising a mixture of pertactins of Bordetella species, wherein saidcomposition comprises: (a) pertactin of Bordetella parapertussis, and(b) pertactin of Bordetella bronchiseptica, in amounts sufficient toinduce a humoral or cellular immune response against Bordetellaparapertussis and Bordetella bronchiseptica in an animal to which theimmunogenic composition is administered. The immunogenic composition canalso comprise pertactin of Bordetella pertussis in an amount sufficientto induce a humoral or cellular immune response against Bordetellapertussis in an animal to which the immunogenic composition isadministered.

[0015] In another embodiment, the immunogenic composition of theinvention comprises a mixture of pertactins of Bordetella species orfragments thereof. Specifically, the mixture comprises a mixture ofBordetella bronchiseptica pertactin variants wherein each Bordetellabronchiseptica pertactin variant comprises 6, 7, 8, or 9 repeating PQPamino acid sequences in Region II thereof. The Bordetella bronchisepticapertactin variants are present in amounts sufficient to induce a humoralor cellular immune response against Bordetella bronchiseptica in ananimal to which the immunogenic composition is administered. Thisimmunogenic composition can also comprise pertactins of Bordetellaparapertussis, Bordetella pertussis, or mixtures thereof, in amountssufficient to induce a humoral or cellular immune response againstBordetella parapertussis or Bordetella pertussis in an animal to whichthe immunogenic composition is administered.

[0016] In a further embodiment of the invention, the immunogeniccomposition comprises a mixture of pertactins of Bordetella species orfragments thereof, wherein mixture comprises a mixture of Bordetellabronchiseptica pertactin variants, wherein each Bordetellabronchiseptica pertactin variant comprises 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof. The Bordetella bronchisepticapertactin variants are present in amounts sufficient to induce a humoralor cellular immune response against Bordetella bronchiseptica in ananimal to which the immunogenic composition is administered. Thisimmunogenic composition can also comprise pertactins of Bordetellaparapertussis, Bordetella pertussis, or mixtures thereof, in amountssufficient to induce a humoral or cellular immune response againstBordetella parapertussis or Bordetella pertussis in an animal to whichthe immunogenic composition is administered.

[0017] The compositions of the invention can comprise a mixture offragments of the pertactins of Bordetella species. The immunogeniccompositions can also comprise at least one polypeptide of the inventionin an amount sufficient to induce an immunogenic or protective responsein vivo, and a pharmaceutically acceptable carrier therefor. Inaddition, the immunogenic composition can comprise a neutralizing amountof at least one polypeptide of the invention.

[0018] A preferred immunogenic composition of this invention comprises amixture of pertactins of Bordetella bronchiseptica species or fragmentsthereof, wherein the pertactins or fragments thereof comprise a mixtureof Bordetella bronchiseptica pertactin variants in which at least one ofthe Bordetella bronchiseptica pertactin variants comprises Region II ofpertactin of Bordetella bronchiseptica having 6, 7, 8, or 9 repeatingPQP amino acid sequences in Region II thereof, and at least another ofthe Bordetella bronchiseptica pertactin variants comprises Region I ofpertactin of Bordetella bronchiseptica having 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof.

[0019] In another preferred embodiment, the immunogenic composition ofthe invention consists essentially of (A) a polypeptide comprisingRegion I and Region II, or one polypeptide comprising Region I and onepolypeptide comprising Region II, of a pertactin of Bordetellapertussis; (B) a polypeptide comprising Region I and Region II, or onepolypeptide comprising Region I and one polypeptide comprising RegionII, of a pertactin of Bordetella parapertussis; (C) a polypeptidecomprising Region I and Region II, or one polypeptide comprising RegionI and one polypeptide comprising Region II, of a pertactin of Bordetellabronchiseptica strain 9.73 and a polypeptide comprising Region I andRegion II, or one polypeptide comprising Region I and one polypeptidecomprising Region II, of a pertactin of Bordetella bronchiseptica ofstrain SEI.

[0020] This invention also provides polynucleotides encoding theproteins and polypeptides of the invention, as well as antibodies thatrecognize the proteins and polypeptides. Also provided is a DNA chip,wherein said chip comprises at least one polynucleotide according to theinvention or fragment thereof or a microarray comprising microbeads,wherein the microbeads each bears multiple copies of a polynucleotideaccording to claims 28-31 or a fragment thereof and wherein thepolynucleotide or fragment thereof is different from one bead toanother.

[0021] The antibodies can be monoclonal or polyclonal antibodies.Monoclonal antibodies can be used for treating Bordetella infections.Also provided are immunological complexes comprising a protein orpolypeptide of the invention and an antibody that specificallyrecognizes the protein or polypeptide.

[0022] Further, this invention provides a method for detecting infectionby Bordetella. The method comprises providing a composition comprising abiological material suspected of being infected with Bordetella andassaying for the presence of a protein or polypeptide of the invention.The polypeptide can be assayed, for example, by electrophoresis or byimmunoassay with antibodies that are immunologically reactive with thepolypeptide.

[0023] The method can also comprise contacting the antigen with abiological fluid for a time and under conditions sufficient for theantigen and antibodies in the biological fluid to form anantigen-antibody complex, and detecting the formation of the complex.The method optionally can include measuring the formation of theantigen-antibody complex. In preferred embodiments, formation ofantigen-antibody complex is detected by immunoassay based on Westernblot technique, ELISA, indirect immunofluorescence assay, orimmunoprecipitation assay.

[0024] Further, this invention provides a diagnostic kit for thedetection of the presence or absence of antibodies, which bind a proteinor polypeptide of the invention or mixtures thereof. The kit cancomprise an antigen comprising the protein or polypeptide, or mixturesof the proteins and polypeptides, and means for detecting the formationof immune complexes between the antigen and antibodies. The means arepresent in an amount sufficient to perform the detection.

[0025] Another method of the invention for detecting the presence orabsence of Bordetella comprises (1) contacting a sample suspected ofcontaining genetic material of Bordetella with at least one nucleotideprobe, and (2) detecting hybridization between the nucleotide probe andthe genetic material in the sample. The nucleotide probe iscomplementary to a polynucleotide sequence of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] This invention will be described in greater detail with referenceto the drawings in which:

[0027]FIG. 1a is a map of the two regions of repeats, Region I andRegion II, in the pertactin outer membrane protein of Bordetellabronchiseptica.

[0028]FIG. 1b is an alignment of Region I of the pertactin outermembrane protein of different strains of B. bronchiseptica.

[0029]FIG. 1c is an alignment of Region II of the pertactin outermembrane protein of different strains of B. bronchiseptica.

DETAILED DESCRIPTION OF THE INVENTION

[0030] It has been demonstrated previously that species-specific membersof the pertactin family are outer-membrane proteins (OMPs). In B.bronchiseptica, pertactin is the product of the pm gene and isrepresented as a protein with an M_(r) of 68 kDa (P.68), in B. pertussisas a protein with an M _(r) of 69 kDa (P.69), and in B. parapertussis asa protein with an M_(r) of 70 kDa (P.70). The nucleotide sequences ofthe pertactins of these three species are included in the accompanyingSequence Listing as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3,respectively. The corresponding amino acid sequences encoded by thesenucleotide sequences are included in the sequence listing as SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0031] A comparison of the deduced protein sequences for the P.68, P.69.and P.70 proteins demonstrates the high degree of homology between theproteins. A comparison between the P.68 and P.70 proteins shows only 17amino acid differences, while a similar comparison between P.68 and P.69shows 80 differences, and 79 differences between P.69 and P.70. Themajority of amino acid differences between the three deduced proteinsequences occur in the number of repeat units in the two families ofrepeat sequences present in all three proteins. P.68 has three copies ofthe Gly-Gly-Xaa-Xaa-Pro repeat (i.e., GGXXP in FIG. 1b), while P.70 hasfour and P.69 five. Similarly, P.68 has seven Pro-Gln-Pro repeats (i.e.,PQP in FIG. 1c), P.70 has nine and P.69 has five.

[0032] It has recently been shown that the PRN produced by clinicalisolates of B. pertussis varies. Sequences of the prn gene of variousclinical isolates revealed three major types of PRN variant. It has beensuggested that epidemics in the Netherlands result from changes in thesequences of the genes encoding PRN and PT because the proteins presentin the clinical isolates currently in circulation differ in sequencefrom those observed by the vaccinal strains used in this country.

[0033] An aim of the searches, which led to the present invention, wasto analyze whether the PRN polymorphism observed in B. pertussis speciesalso occurs in B. parapertussis and B. bronchiseptica. The two repeatedregions of the prn genes of 10 B. parapertussis isolates of human originand of 40 B. bronchiseptica isolates of animal or human origin weresequenced and compared. (FIG. 1a). TABLE I PRN regions I AccessionBordetella Representative and II types/ number,* Species isolate Numberof isolates region I, region II BB 9.73H+ I-1, II-3/3  AJ250076,AJ250077 BB LAPR I-2, II-3/8  AJ250078, AJ250079 BB 5 1-2, II-4/8 AJ250080, AJ250081 BB 335 I-2, II-1/3  AJ250082, AJ250083 BB CVGEO I-2,II-5/6  AJ250084, AJ250085 BB BBCH I-2, II-6/4  AJ250086, AJ250087 BBDEL I-1, II-2/5  AJ250088, AJ25089 BB CAT1 I-1, II-7/1  AJ250090,AJ250091 BB 286 I-3, II-8/1  AJ250093, AJ250092 BB SEI I-3, II-9/1 AJ250094, AJ250095 BPP 63.2  I-1, II-2/10 Identical to P24328 SpeciesStrain PRN type Accession number BPP CN2591 I-1, II-2 P24328 BB CN7531I-2, II-4 Q03035 Species Strain or isolate Allelic prn type Accessionnumber BP Tohama prn1 AJ006158 BP 18323 prn6 AJ006152 BP Hav prn2AJ007361 BP Fr287 prn3 AJ006156

[0034] In carrying out this invention, DNA was extracted, amplified byPCR, and sequenced, as previously described (3). Amplified PCR productswere purified and sequenced by the ESGS company (ESGS, Cybergene group,Evry, France). Deduced amino acid sequences were analyzed with GCGsoftware (Wisconsin Package Version 9. 1, Genetics Computer Group,Madison, Wis., USA). The deduced amino acid sequences of regions I andII were compared and multiple alignments of the amino acid sequenceswere created with the CLUSTAL W program of GCG (10), for each region(FIG. 1b,c).

[0035] No difference was found between the sequences of regions I and IIof the PRN produced by the 10 B. parapertussis isolates and thepublished sequence (15). However, three different types were found amongthe 40 B. bronchiseptica prn genes analyzed with differences in thenumber of repeats (1 to 3) in region I (FIG. 1b). The largest groupcorresponded to sequences with three copies of the repeated sequence,identical to the sequence previously reported (14). No correlation wasfound between the pattern of variation and the origin of the isolate.

[0036] A higher degree of variability was observed in the secondrepeated region of the B. bronchiseptica PRN (FIG. 1c). Nine variantswere observed. Among these nine variants the number of repeats is from 6to 9.

[0037] No B. bronchiseptica variants presented the same pattern as theB. pertussis variants. Furthermore, no unique association between onetype of region I and one type of region II was observed. No observationwas made in any of the three species of a pattern similar to those ofthe 18323 strain and the CZ isolate (3), which are considered to beintermediate between B. pertussis, B. bronchiseptica, and parapertussis.These data are consistent with B. parapertussis and B. bronchisepticaprn genes being more similar to each other than to the B. pertussisprngene (1). No host specificity was observed with respect to PRN type.

[0038] It has been shown that region II plays an important role in theinduction of protective immunity (6). The lack of cross-protectionbetween PRN from B. pertussis, B. parapertussis, and B. bronchisepticaPRN is consistent with this, because the major differences between theseproteins occur in this region. No variation in this region was observedfor the PRN produced by B. pertussis isolates. These data suggest thatthirty years of vaccination may have induced variation in oneimmunodominant repeat region, but not in the region most involved in theinduction of protective immunity. Variation in B. pertussis PRN regionII may indicate a decrease in B. pertussis vaccine efficacy.

[0039] In contrast, analysis of the PRN of B. bronchiseptica showedpolymorphism in both regions. This may account for the inability of B.bronchiseptica vaccines to induce long-lasting protection. Thispolymorphism may also be linked to the ability of B. bronchiseptica toinduce chronic infections (7, 8, 22). It may provide a means for thisbacterium to escape host immune responses.

[0040] This invention, which resulted from these experiments andobservations, thus involves compositions containing certain Bordetellapertactins and fragments thereof. These pertactins and pertactinfragments, as well as the polynucleotides that encode them, are usefulin immunogenic compositions and in diagnostic applications.

[0041] In particular, this invention is the result of the discovery thatthere are different species of the full length pertactin of Bordetellabronchiseptica, namely, species containing 6, 7, 8, or 9 repeating PQPamino acid sequences in Region II thereof, and species of full lengthpertactin of B. bronchiseptica containing 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof, where XX can be FD, FG, or AV.These fall length pertactins and mixtures of these pertactins in anycombination of the repeating sequences are thus provided by thisinvention.

[0042] As used herein, the expression “pertactin of Bordetellabronchiseptica” means an outer membrane protein of Bordetellabronchiseptica, which is a virulence factor, and which has an apparentmolecular weight of about 68 kDa, and which contains the two regions ofBordetella bronchiseptica pertactin known as Region I and Region II.Region I and Region II of the pertactins of different Bordetella strainsare identified in brackets in SEQ ID NOS: 1 to 6. It will be understoodthat the pertactins of different isolates of Bordetella bronchisepticamay have amino acid sequences that differ from each other, for example,in Region I, Region II, or both Region I and Region II, as well as inother regions.

[0043] As used herein the expression “Bordetella bronchisepticapertactin variants” means pertactins of Bordetella bronchiseptica, orfragments of pertactins of Bordetella bronchiseptica containing at leastRegion I, Region II, or both Region I and Region II, in which thepertactins of Bordetella bronchiseptica or the fragments thereof differfrom each other in at least Region I, Region II, or both Region I andRegion II, in their respective amino acid sequences. The followingunique Bordetella bronchiseptica pertactin variants have been discoveredand constitute part of this invention.

[0044] As used herein the expressions Bordetella bronchisepticapertactin fragments”, “Bordetella parapertussis pertactin fragments”,and “Bordetella pertussis pertactin fragments” refer to polypeptidesthat are portions of full length pertactin proteins and are capable ofinducing a humoral or immune response against Bordetella infections. B.bronchiseptica pertactin-region I I-1QRATIRRGDAPAGGAVPGGAVPGGAVPG---------------GFGPLLDGWYGVDVSDSTVDLAQ (SEQID NO:7) I-2QRATIRRGDAPAGGAVPG-----GAVPG---------------GFGPLLDGWYGVDVSDSTVDLAQ (SEQID NO:8) I-3QRATIRRGDAPAGGGVPG-----GAVPG-----GFDPGGFGPGGFGPVLDGWYGVDVSGSTVELAQ (SEQID NO:9) prn1QRATIRRGDAPAGGAVPG-----GAVPG-----GAVPGGFGPGGFGPVLDGWYGVDVSGSSVELAQ (SEQID NO:10) prn2QRATIRRGDAPAGGAVPG-----GAVPGGFGPGGFGPGGFGPGGFGPVLDGWYGVDVSGSSVELAQ (SEQID NO:11) prn3QRATIRRGDAPAGGAVPG-----GAVPG-----GFGPGGFGPGGFGPVLDGWYGVDVSGSSVELAQ (SEQID NO:12) prn4QRATIRRGDAPAGGAVPG-----GAVPG----------GFGPGGFGPVLDGWYGVDVSGSSVELAQ (SEQID NO:13)  **************.***     ****                ****:*********.*:*:***B.bronchiseptica pertactin-region II II-1GAKAPPAPKPAPQPGPQPGP-----------QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO:14) II-2GAKAPPAPKPAPQPGPQPGPQPP--------QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO:15) II-3GAKAPPAPKPAPQPGPQPGPQPGPQPGPQPPQPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO:16) II-4GAKAPPAPKPAPQPGPQPGPQPGP-------QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO:17) II-5GAKAPPAPKPAPQPGPQPGPQPGPQP----PQPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO:18) II-6GAKAPPAPKPAPQPGPQPGPQPPQPP--QPPQPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO:19) II-7GAKAPPAPKPAPQPGPQP-P-----------QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO:20) II-8GAKVPPAPKPAPQPGPQP-PQPP--------QPPQPPQPQPQPQP--EAPAPQPPAGRELSAA (SEQ IDNO:21) II-9GAKVPPAPKPAPQPGPQP-PQPP--------QPPQPPQPQPQPQPQPEAPAPQPPAGRELSAA (SEQ IDNO:22) prnlGAKAPPAPKPAPQPGPQP---------------PQPPQP----QP--EAPAPQPPAGRELSAA (SEQ IDNO:23) prn6GAKAPPAPKPAPQPGPQP------------------PQP----QP--EAPAPQPPAGRELSAA (SEQ IDNO:24)

[0045] In specific embodiments, this invention includes a polypeptidecomprising a sequence or a fragment of a sequence selected from thegroup consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22. The polypeptide can consistof the amino acids in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22 or fragments thereof.The invention also includes polynucleotides encoding one of thesepolypeptides and a purified DNA or RNA sequence that hybridizes undermoderate or high stringency conditions to the polynucleotides or atleast to 15 nucleotides thereof.

[0046] As used herein, the expression “mixture of Bordetellabronchiseptica pertactin variants” means two or more Bordetellabronchiseptica pertactin variants in admixture in solid, liquid,emulsion, or suspension form. At least two of the Bordetellabronchiseptica pertactin variants in the mixture will, of course, differfrom each other in at least Region I, Region II, or both Region I andRegion II, in their respective amino acid sequences.

[0047] It will be immediately apparent that this invention providespolypeptide fragments of the pertactin of B. bronchiseptica, where thefragments comprise 6, 7, 8, or 9 repeating PQP amino acid sequences inRegion II thereof or 1, 2, or 3 repeating GGXXP amino acid sequences inRegion I thereof. Mixtures of these polypeptide fragments in anycombination of the repeating sequences are also within the scope of thisinvention.

[0048] When a polypeptide fragment of the invention comprises onlyRegion I of a pertactin of B. bronchiseptica, the polypeptide fragmenttypically contains at least about 46 to about 56 amino acids, whichincludes the Region I repeat sequences. When the polypeptide fragment ofthe invention comprises only Region II, the polypeptide fragmenttypically contains at least about 48 to about 60 amino acids, whichincludes the Region II repeat sequences. When the polypeptide fragmentof the invention comprises both Region I and Region II of B.bronchiseptica, the fragment typically contains at least about 906 toabout 928 amino acids, which includes the repeat sequences of Regions Iand II.

[0049] Thus, in one illustrative embodiment, this invention provides acomposition comprising a mixture of Bordetella bronchiseptica pertactinvariants, wherein each Bordetella bronchiseptica pertactin variantcomprises Region II of pertactin of Bordetella bronchiseptica, andfurther wherein each Bordetella bronchiseptica pertactin variantcomprises 6, 7, 8, or 9 repeating PQP amino acid sequences in Region IIthereof, and the Bordetella bronchiseptica pertactin variants differ inthe number of the repeating PQP amino acid sequences contained therein.The composition can also comprise pertactins of Bordetellaparapertussis, Bordetella pertussis, or mixtures thereof. Thepolypeptide can be a full length pertactin or a fragment thereof.

[0050] In another embodiment, this invention provides a compositioncomprising a mixture of Bordetella bronchiseptica pertactin variants,wherein each Bordetella bronchiseptica pertactin variant comprisesRegion I of a pertactin of Bordetella bronchiseptica, and furtherwherein each Bordetella bronchiseptica pertactin variant comprises 1, 2,or 3 repeating GGXXP amino acid sequences in Region I thereof, and theat least two of the Bordetella bronchiseptica pertactin variants differin the number of the repeating GGXXP amino acid sequences containedtherein. This composition can also comprise pertactins of Bordetellaparapertussis, Bordetella pertussis, or mixtures thereof. The Bordetellabronchiseptica pertactin variants can be full length or a fragment.

[0051] In a further embodiment, the invention provides a compositioncomprising a mixture of Bordetella bronchiseptica pertactin variants,wherein one of the Bordetella bronchiseptica pertactin variantscomprises Region II of pertactin of Bordetella bronchiseptica having 6,7, 8, or 9 repeating PQP amino acid sequences in Region II thereof, andanother of the Bordetella bronchiseptica pertactin variants comprisesRegion I of pertactin of Bordetella bronchiseptica having 1, 2, or 3repeating GGXXP amino acid sequences in Region I thereof. Thiscomposition can also comprise pertactins of Bordetella parapertussis,Bordetella pertussis, or mixtures thereof. The Bordetella bronchisepticapertactin variants can be full length or a fragment.

[0052] In a preferred embodiment, this invention provides a polypeptidecomprising a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0053] In another preferred embodiment, this invention provides apolypeptide comprising a sequence selected from the group consisting ofSEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0054] The compositions according to the invention cause a humoralimmune response and a cellular immune response. After infection with B.bronchiseptica, there is induction of a humoral immunity and of acellular immunity, as in the case of a B. pertussis and B. parapertussisinfection. Furthermore, after vaccination with compositions of thisinvention, there is induction of a humoral and cellular type immunitysimilar to that induced after infection or reinfection.

[0055] In one embodiment of the invention there is provided avaccinating composition comprising as active principle an immunogeniccomposition of the invention, in combination with a pharmaceuticallyacceptable vehicle and, where appropriate, with an adjuvant.

[0056] Like the whooping cough vaccines currently available on themarket, the immunogenic composition according to the invention may becombined with other vaccinating active principles, for example, those ofthe vaccine against diphtheria, polio, or diseases caused by Haemophilusor, generally speaking, with any immunogenic constituent, for example, aparticular inactivated pathogenic agent or toxin.

[0057] A vaccinating composition according to the invention can bespecies-specific and consequently capable of inducing protection againstB. pertussis or B. parapertussis or B. bronchiseptica. Alternatively, itcan be a mixture comprising as active principle an immunogeniccomposition against B. bronchiseptica, as defined above, and animmunogenic composition against B. parapertussis and/or B. pertussis.

[0058] As a result of recent techniques in molecular biology, a numberof factors involved in the virulence of B. pertussis have beencharacterized and the regulation of their expression understood. Thesefactors may be classified in two categories, those participating in theinfectious syndrome (adhesins) and those playing a part in thetoxin-induced syndrome (toxins). The adhesins and toxins relating toBordetella can be included in the compositions of this invention.Examples of the adhesins are:

[0059] filamentous hemagglutinin or FHA, considered to play a major partin the adhesion of the bacterium to the ciliated epithelium;

[0060] the two agglutinogens or AGGs of B. pertussis, which enablestrains to be classified in serotypes; and

[0061] pertussis toxin or PTX, a secreted type A-B toxin which, besidesits cytopathogenic effects, participates in adhesion via its B subunit.

[0062] Examples of the toxins for use in the invention are:

[0063] pertussis toxin or PTX, which is secreted;

[0064] dermonecrotic toxin or DNT, which function has not yet been wellcharacterized, and tracheal cytotoxin or TCT, a secreted smallglycoprotein of the muramyl peptide family, derived from thepeptidoglycan of the bacterium, which appear to act in concert todestroy the ciliated cells of the host's respiratory apparatus;

[0065] adenylate cyclase-hemolysin or Ac-Hly, a bifunctional proteinpossessing adenylate cyclase activity and hemolytic activity, which hasbeen found to belong to the family of toxins termed “RTX” for “repeatsin toxins”.

[0066] Similarly, the factors involved in the virulence of B.parapertussis and B. bronchiseptica have been identified and can beincluded in the compositions of the invention.

[0067] The published results show that the acellular vaccines tested,monovalent (PTX), bivalent (PTX, FHA), trivalent (PTX, FHA, PRN), orpentavalent (PTX, FHA, PRN, AGG2, AGG3) induce very few side effects,are all immunogenic and all have an efficacy against the disease(according to WHO definition) which is greater than or equal to 70%. Thecompositions of the invention can be included in these vaccines andother acellular vaccines. For example, the immunogenic composition canfurther comprise at least one adhesin of Bordetella selected from thegroup consisting of FHA, AGG2, AGG3, and/or at least one toxin ofBordetella selected from the group consisting of PTX, DNT, TCT, andAc-Hly.

[0068] The proteins, polypeptides, and compositions of this inventioncan be in purified form. The term “purified” as used herein, means thatthe pertactins and fragments thereof are essentially free of associationwith other proteins or polypeptides, for example, as a purificationproduct of recombinant host cell culture or as a purified product from anon-recombinant source. The term “substantially purified” as usedherein, refers to a mixture that contains pertactins or fragmentsthereof and is essentially free of association with other proteins orpolypeptides, but for the presence of known proteins that can be removedusing a specific antibody, and which substantially purified pertactinpolypeptides can be used as antigens.

[0069] Within an aspect of the invention, the pertactin and fragmentsthereof can be utilized to prepare antibodies that specifically bind topertactin polypeptides. The term “antibodies” is meant to includepolyclonal antibodies, monoclonal antibodies, fragments thereof, such asF(ab′)2 and Fab fragments, as well as any recombinantly produced bindingpartners. Antibodies are defined to be specifically binding if they bindpertactins and fragments thereof with a K_(a) of greater than or equalto about 10⁷ M⁻¹. Affinities of binding partners or antibodies can bereadily determined using conventional techniques, for example, thosedescribed by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice, or rats, using procedures that are well known in the art.

[0070] The invention further encompasses isolated fragments andoligonucleotides derived from the nucleotide sequence of the pertactinsB. bronchiseptica, B. pertussis and B. parapertussis (SEQ ID NO:1, SEQID NO:2, and SEQ ID NO:3) encoding 6, 7, 8, or 9 repeating PQP aminoacid sequences in Region II thereof, and/or 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof. The invention also encompassespolypeptides encoded by these fragments and oligonucleotides. Mixturescan comprise nucleotide sequences containing repeating sequences inwhich each entity in the mixture is independently selected from thepolynucleotides of the invention.

[0071] Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the native pertactinnucleic acids disclosed herein under conditions of moderate or severestringency, and which encode pertactin polypeptides. As used herein,conditions of moderate stringency, as known to those having ordinaryskill in the art, and as defined by Sambrook et al. Molecular Cloning: ALaboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring HarborLaboratory Press, (1989), include use of a prewashing solution for thenitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization conditions of 50% formamide, 6×SSC at 42° C. (or othersimilar hybridization solution, such as Stark's solution, in 50%formamide at 42° C.), and washing conditions of about 60° C., 0.5×SSC,0.1% SDS. Conditions of high stringency are defined as hybridizationconditions as above, and with washing at 68° C., 0.2×SSC, 0. 1% SDS. Theskilled artisan will recognize that the temperature and wash solutionsalt concentration can be adjusted as necessary according to factorssuch as the length of the probe.

[0072] Due to the known degeneracy of the genetic code, wherein morethan one codon can encode the same amino acid, a DNA sequence can varyand still encode a pertactin polypeptide having the amino acid sequenceof SEQ ID NO:7 through SEQ ID NO:24. Such variant DNA sequences canresult from silent mutations (e.g., occurring during PCR amplification),or can be the product of deliberate mutagenesis of a native sequence.

[0073] The invention thus provides equivalent isolated DNA sequences,encoding pertactin polypeptides, selected from: (a) DNA derived from thecoding region of a native pertactin gene; (b) cDNA comprising thenucleotide sequence of SEQ ID NO:7 through SEQ ID NO:24; (c) DNA capableof hybridization to a DNA of (a) under conditions of moderate stringencyand which encode pertactin polypeptides; and (d) DNA which is degenerateas a result of the genetic code to a DNA defined in (a), (b) or (c) andwhich encodes pertactin polypeptides. Pertactin polypeptides encoded bysuch DNA equivalent sequences are encompassed by the invention.

[0074] It will be understood that the present invention is intended toencompass the previously described proteins and polypeptides in isolatedor purified form, whether obtained using the techniques described hereinor other methods. In a preferred embodiment of this invention, thepertactin polypeptides are substantially free of human or other animaltissue and human or other animal tissue components, nucleic acids,extraneous proteins and lipids, and adventitious microorganisms, such asbacteria and viruses. It will also be understood that the inventionencompasses equivalent proteins having substantially the same biologicaland immunogenic properties. Thus, this invention is intended to coverserotypic variants of the polypeptides of the invention.

[0075] Depending on the use to be made of the pertactin polypeptides ofthe invention, it may be desirable to label them. Examples of suitablelabels are radioactive labels, enzymatic labels, fluorescent labels,chemiluminescent labels, and chromophores. The methods for labeling donot differ in essence from those widely used for labelingimmunoglobulin. The need to label may be avoided by using labeledantibody to the antigen of the invention or anti-immunoglobulin to theantibodies to the antigen as an indirect marker.

[0076] Once the pertactin polypeptides of the invention have beenobtained, they can be used to produce polyclonal and monoclonalantibodies reactive therewith. Thus, a protein or polypeptide of theinvention can be used to immunize an animal host by techniques known inthe art. Such techniques usually involve inoculation, but they mayinvolve other modes of administration. A sufficient amount of theprotein or the polypeptide is administered to create an immunogenicresponse in the animal host. Any host that produces antibodies to theantigen of the invention can be used. Once the animal has been immunizedand sufficient time has passed for it to begin producing antibodies tothe antigen, polyclonal antibodies can be recovered. The general methodcomprises removing blood from the animal and separating the serum fromthe blood. The serum, which contains antibodies to the antigen, can beused as an antiserum to the antigen. Alternatively, the antibodies canbe recovered from the serum. Affinity purification is a preferredtechnique for recovering purified polyclonal antibodies to the antigen,from the serum.

[0077] Monoclonal antibodies to the antigens of the invention can alsobe prepared. One method for producing monoclonal antibodies reactivewith the antigens comprises the steps of immunizing a host with theantigen; recovering antibody producing cells from the spleen of thehost; fusing the antibody producing cells with myeloma cells deficientin the enzyme hypoxanthine-guanine phosphoribosyl transferase to formhybridomas; select at least one of the hybridomas by growth in a mediumcomprising hypoxanthine, aminopterin, and thymidine; identifying atleast one of the hybridomas that produces an antibody to the antigen,culturing the identified hybridoma to produce antibody in a recoverablequantity; and recovering the antibodies produced by the culturedhybridoma.

[0078] These polyclonal or monoclonal antibodies can be used in avariety of applications. Among these is the neutralization ofcorresponding proteins. They can also be used to detect Bordetellaantigens in biological preparations or in purifying correspondingproteins, glycoproteins, or mixtures thereof, for example when used in aaffinity chromatographic columns.

[0079] The pertactin polypeptides of the invention can be used asantigens to identify antibodies to Bordetella in materials and todetermine the concentration of the antibodies in those materials. Thus,the antigens can be used for qualitative or quantitative determinationof Bordetella in a material. Such materials, of course, include human orother animal tissue and human or other animal cells, as well asbiological fluids, such as human or other animal body fluids, includinghuman sera. When used as a reagent in an immunoassay for determining thepresence or concentration of the antibodies to Bordetella, the antigensof the present invention provide an assay that is convenient, rapid,sensitive, and specific.

[0080] More particularly, the antigens of the invention can be employedfor the detection of Bordetella by means of immunoassays that are wellknown for use in detecting or quantifying humoral components in fluids.Thus, antigen-antibody interactions can be directly observed ordetermined by secondary reactions, such as precipitation oragglutination. In addition, immunoelectrophoresis techniques can also beemployed. For example, the classic combination of electrophoresis inagar followed by reaction with anti-serum can be utilized, as well astwo-dimensional electrophoresis, rocket electrophoresis, andimmunolabeling of polyacrylamide gel patterns (Western Blot orimmunoblot.) Other immunoassays in which the antigens of the presentinvention can be employed include, but are not limited to,radioimmunoassay, competitive immunoprecipitation assay, enzymeimmunoassay, and immunofluorescence assay. It will be understood thatturbidimetric, colorimetric, and nephelometric techniques can beemployed. An immunoassay based on Western Blot technique is preferred.

[0081] Immunoassays can be carried out by immobilizing one of theimmunoreagents, either an antigen of the invention or an antibody of theinvention to the antigen, on a carrier surface while retainingimmunoreactivity of the reagent. The reciprocal immunoreagent can beunlabeled or labeled in such a manner that immunoreactivity is alsoretained. These techniques are especially suitable for use in enzymeimmunoassays, such as enzyme linked immunosorbent assay (ELISA) andcompetitive inhibition enzyme immunoassay (CIEIA).

[0082] When either the antigen of the invention or antibody to theantigen is attached to a solid support, the support is usually a glassor plastic material. Plastic materials molded in the form of plates,tubes, beads, or disks are preferred. Examples of suitable plasticmaterials are polystyrene and polyvinyl chloride. If the immunoreagentdoes not readily bind to the solid support, a carrier material can beinterposed between the reagent and the support. Examples of suitablecarrier materials are proteins, such as bovine serum albumin, orchemical reagents, such as gluteraldehyde or urea. Coating of the solidphase can be carried out using conventional techniques.

[0083] The invention provides immunogenic pertactin polypeptides, andmore particularly, protective polypeptides for use in the preparation ofvaccine compositions against Bordetella. These polypeptides can thus beemployed as vaccines by administering the polypeptides to a mammalsusceptible to Bordetella infection. Conventional modes ofadministration can be employed. For example, administration can becarried out by oral, respiratory, or parenteral routes. Intradermal,subcutaneous, and intramuscular routes of administration are preferredwhen the vaccine is administered parenterally.

[0084] The major purpose of the immune response in a Bordetella-infectedmammal is to inactivate the Bordetella and to eliminate Bordetellainfected cells that have the potential to release infectious virus. TheB-cell arm of the immune response has the major responsibility forinactivating Bordetella. The principal manner in which this is achievedis by neutralization of infectivity. Another major mechanism fordestruction of the Bordetella-infected cells is provided by cytotoxic Tlymphocytes (CTL) that recognize pertactin antigens expressed incombination with class I histocompatibility antigens at the cellsurface. The CTLs recognize pertactin polypeptides processed withincells from a pertactin protein that is produced, for example, by theinfected cell or that is internalized by a phagocytic cell. Thus, thisinvention can be employed to stimulate a B-cell response to pertactinpolypeptides, as well as immunity mediated by a CTL response followinginfection. The CTL response can play an important role in mediatingrecovery from primary Bordetella infection and in accelerating recoveryduring subsequent infections.

[0085] The ability of the pertactin polypeptides and vaccines of theinvention to induce protective levels of neutralizing antibody in a hostcan be enhanced by emulsification with an adjuvant, incorporating in aliposome, coupling to a suitable carrier, or by combinations of thesetechniques. For example, the pertactin polypeptides of the invention canbe administered with a conventional adjuvant, such as aluminum phosphateand aluminum hydroxide gel, in an amount sufficient to potentiatehumoral or cell-mediated immune response in the host. Similarly, thepertactin polypeptides can be bound to lipid membranes or incorporatedin lipid membranes to form liposomes. The use of nonpyrogenic lipidsfree of nucleic acids and other extraneous matter can be employed forthis purpose.

[0086] The immunization schedule will depend upon several factors, suchas the susceptibility of the host to infection and the age of the host.A single dose of the vaccine of the invention can be administered to thehost or a primary course of immunization can be followed in whichseveral doses at intervals of time are administered. Subsequent dosesused as boosters can be administered as need following the primarycourse.

[0087] The pertactin proteins, polypeptides, and vaccines of theinvention can be administered to the host in an amount sufficient toprevent or inhibit Bordetella infection or replication in vivo. In anyevent, the amount administered should be at least sufficient to protectthe host against substantial immunosuppression, even though Bordetellainfection may not be entirely prevented. An immunogenic response can beobtained by administering the proteins or polypeptides of the inventionto the host in an amount of, for example, about 1 to about 50 microgramsantigen per kilogram of body weight, preferably about 5 to about 10micrograms antigen per kilogram of body weight. The proteins,polypeptides, and vaccines of the invention can be administered togetherwith a physiologically acceptable carrier. For example, a diluent, suchas water or a saline solution, can be employed.

[0088] Another aspect of the invention includes administering anycombination of the nucleic acids encoding pertactin polypeptides, theproteins, and polypeptides per se, with or without carrier molecules, toan individual. The individual can be an animal. As used herein, the term“animal” means a mammal, and preferably, the mammal is selected from thegroup consisting of a human, a rabbit, a mouse, a dog, a cat, a bovine,a pig, and a horse. In an especially preferred embodiment, the mammal isa human.

[0089] The methods of treating include administering immunogeniccompositions comprising pertactin proteins or polypeptides, andcompositions comprising nucleic acids encoding pertactin proteins orpolypeptides as well. Those of skill in the art are cognizant of theconcept, application, and effectiveness of nucleic acid vaccines (e.g.,DNA vaccines) and nucleic acid vaccine technology as well as protein andpolypeptide based technologies. The nucleic acid based technology allowsthe administration of nucleic acids encoding pertactin polypeptides,naked or encapsulated, directly to tissues and cells without the needfor production of encoded proteins prior to administration. Thetechnology is based on the ability of these nucleic acids to be taken upby cells of the recipient organism and expressed to produce animmunogenic determinant to which the recipient's immune system responds.Typically, the expressed antigens are displayed on the surface of cellsthat have taken up and expressed the nucleic acids, but expression andexport of the encoded antigens into the circulatory system of therecipient individual is also within the scope of the present invention.Such nucleic acid vaccine technology includes, but is not limited to,delivery of naked DNA and RNA and delivery of expression vectorsencoding pertactin polypeptides. Although the technology is termed“vaccine”, it is equally applicable to immunogenic compositions that donot result in a protective response. Such non-protection inducingcompositions and methods are encompassed within the present invention.

[0090] Although it is within the present invention to deliver nucleicacids encoding pertactin polypeptides and carrier molecules as nakednucleic acid, the present invention also encompasses delivery of nucleicacids as part of larger or more complex compositions. Included amongthese delivery systems are viruses, virus-like particles, or bacteriacontaining the nucleic acid encoding pertactin polypeptides. Also,complexes of the invention's nucleic acids and carrier molecules withcell permeabilizing compounds, such as liposomes, are included withinthe scope of the invention. Other compounds, such as molecular vectors(EP 696,191, Samain et al.) and delivery systems for nucleic acidvaccines are known to the skilled artisan and exemplified in, forexample, WO 93 06223 and WO 90 11092, U.S. Pat Nos. 5,580,859, and5,589,466 (Vical patents), which are incorporated by reference herein,and can be made and used without undue or excessive experimentation.

[0091] To further achieve the objects and in accordance with thepurposes of the present invention, a kit capable of diagnosing aBordetella infection is described. This kit, in one embodiment, containsthe DNA sequences of this invention, which are capable of hybridizing tobacterial RNA or analogous DNA sequences to indicate the presence of aBordetella infection. Different diagnostic techniques can be used whichinclude, but are not limited to: (1) Southern blot procedures toidentify cellular DNA which may or may not be digested with restrictionenzymes; (2) Northern blot techniques to identify RNA extracted fromcells; and (3) dot blot techniques, i.e., direct filtration of thesample through a membrane, such as nitrocellulose or nylon, withoutprevious separation on agarose gel. Suitable material for dot blottechnique could be obtained from body fluids including, but not limitedto, serum and plasma, supernatants from culture cells, or cytoplasmicextracts obtained after cell lysis and removal of membranes and nucleiof the cells by centrifugation.

[0092] Following are references of the strains used in the searchconcerning the present invention:

[0093] 9.73H+5, DEL, SEI: Infect Immun. (1993) 61″4072-4078. Gueirard,P. and Guiso, N., filed with CNCM on May 12, 1989, No. 858.

[0094] CVGEO identical to strain CVHAI 286, 335: Microbiol. (1997)143:1433-1441. Le Blay, K. et al.

[0095] 63.2: CIP—Lab. Ident., Inst. Pasteur, Paris, France—J. Clin.Microbiol., 1993, 31, 2745

[0096] TI: CIP81.32—Lab. Ident., Inst. Pasteur, Paris, France—J. Clin.Microbiol., 1993, 31, 2746

[0097] Fr287: Vaccine (1999) 17:2651:2660. Boursaux-Eude, C. et al.

[0098] 18232: ref OMS: ATCC97.97 (CIP63.1).

REFERENCES

[0099] The following references have been cited in this application. Theentire disclosure of each of these references is relied upon andincorporated by reference herein.

[0100] 1. Arico, B., R. Gross, J. Smida, and R. Rappuoli. 1987.Evolutionary relationships in the genus Bordetella. Mol. Microbiol.1:301-308.

[0101] 2. Arico, B., J. F. Miller, C. Roy, S. Stibitz, D. Monack, S.Falkow, R. Gross, and R. Rappuoli. 1989. Sequences required forexpression of Bordetella pertussis virulence factors share homology withprokaryotic signal transduction proteins. Proc. Natl Acad. Sci. USA.86:6671-6675.

[0102] 3. Boursaux-Eude, C., G. Thiberge, G. Carletti, and N. Guiso.1999. Intranasal murine model of Bordetella pertussis infection: II.Sequence variation and protection induced by a tricomponent acellularvaccine. Vaccine. Infect. Immum. 56:3189-3195.

[0103] 4. Brennan, M. J., Z. M. Li, J. L. Cowell, M. E. Bisher, A. C.Steven, P. Novotny, and C. R. Manclark. 1988. Identification of a69-kilodalton nonfimbrial protein as an agglutinogen of Bordetellapertussis. Infect. Immun. 56:3189-3195.

[0104] 5. Charles, I. G., G. Dougan, D. Pickard, S. Chatfield, M. Smith,P. Novotny, P. Morrissey, and N. F. Fairweather. 1989. Molecular cloningand characterization of protective outer membrane protein P.69 fromBordetella pertussis. Proc. Natl. Acad. Sci. USA. 86:3554-3558.

[0105] 6. Charles, I. G., J. L. Li, M. Roberts, K. Beesley, M. Romanos,D. J. Pickard, M. Francis, D. Campbell, G. Dougan, M. J. Brennan, C. R.Manclarck, M. A. Jensen, I. Heron, A. Chubb, P. Novotny, and N. F.Fairweather. 1991. Identification and characterization of a protectiveimmunodominant B cell epitope of pertactin (P.69) from Bordetellapertussis. Eur. J. Immunol. 21:1147-1153.

[0106] 7. Goodnow, R.A. 1980. Biology of Bordetella bronchiseptica.Microbiol. Rev. 44:722-738.

[0107] 8. Gueirard, P., C. Weber, A. Le Coustumier, and N. Guiso. 1995.Human Bordetella bronchiseptica infection related to contact withinfected animals: persistence of bacteria in host. J. Clin. Microbiol.33:2002-2006.

[0108] 9. Hewlett, E. L., and J. D. Cherry. 1997. New and improvedvaccines against pertussis, vol. 2nd. Coordinating eds., M. M. Levine,G. C. Woodrow, J. B. Kaper, and G. S. Cobon. Marcel Dekker, New York.

[0109] 10. Higgins, D. G., and P. M. Sharp. 1988. CLUSTAL: a package forperforming multiple sequence alignment on a microcomputer. Gene.73:237-244.

[0110] 11. Khelef, N., B. Danve, M. J. Quentin-Millet, and N. Guiso.1993. Bordetella pertussis and Bordetella parapertussis: twoimmunologically distinct species. Infect. Immun. 61:486-490.

[0111] 12. Kobisch, M., and P. Novotny. 1990. Identification of a68-kilodalton outer membrane protein as the major protective antigen ofBordetella bronchiseptica by using specific-pathogen-free piglets.Infect. Immun. 58:352-357.

[0112] 13. Leininger, E., M. Roberts, J. G. Kenimer, I. G. Charles, N.Fairweather, P. Novotny, and M. J. Brennan. 1991. Pertactin, anArg-Gly-Asp-containing Bordetella pertussis surface protein thatpromotes adherence of mammalian cells. Proc. Natl. Acad. Sci. USA.88:345-349.

[0113] 14. Li, J., N. F. Fairweather, P. Novotny, G. Dougan, and I. G.Charles. 1992. Cloning, nucleotide sequence and heterologous expressionof the protective outer-membrane protein P.68 pertactin from Bordetellabronchiseptica. J. Gen. Microbiol. 138:1697-1705.

[0114] 15. Li, L. J., G. Dougan, P. Novotny, and I. G. Charles. 1991.P.70 pertactin, an outer-membrane protein from Bordetella parapertussis:cloning, nucleotide sequence and surface expression in Escherichia coli.Mol. Microbiol. 5:409-417.

[0115] 16. Montaraz, J. A., P. Novotny, and J. Ivanyi. 1985.Identification of a 68-kilodalton protective protein antigen fromBordetella bronchiseptica. Infect. Immun. 47:744-751.

[0116] 17. Mooi, F. R., H. van Oirschot, K. Heuvelman, H. G. J. van derHeide, W. Gaastra, and R. J. L. Willems. 1998. Polymorphism in theBordetella pertussis virulence factors P.69/pertactin and pertussisroxin in the Netherlands: Temporal trends and evidence doevaccine-driven evolution. Infect. Immun. 66:670-675.

[0117] 18. Novotny, P., A. P. Chubb, K. Cownley, J. A. Montaraz, and J.E. Beesley. 1985. Bordetella adenylate cyclase: a genus specificprotective antigen and virulence factor. Develp. Biol. Standard.61:27-41.

[0118] 19. Novotny, P., M. Kobisch, K. Cownley, A. P. Chubb, and J. A.Montaraz. 1985. Evaluation of Bordetella bronchiseptica vaccines inspecific-pathogen-free piglets with bacterial cell surface antigens inenzyme-linked immunosorbent assay. Infect. Immun. 50:190-198.

[0119] 20. Shahin, R. D., M. J. Brennan, Z. M. Li, B. D. Meade, and C.R. Manclark. 1990. Characterization of the protective capacity andimmunogenicity of the 69-kD outer membrane protein of Bordetellapertussis. J. Exp. Med. 171:63-73.

[0120] 21. Stibitz, S., W. Aaronson, D. Monack, and S. Falkow. 1989.Phase variation in Bordetella pertussis by frameshift mutation in a genefor a novel two-component system. Nature. 338:266-269.

[0121] 22. Woolfrey, B. F., and J. A. Moody. 1991. Human infectionsassociated with Bordetella bronchiseptica. Clin. Microbiol. Rev.4:243-255. B. bronchiseptica p.68 pertactin gene [SEQ ID NO:1]atcgatgatg cgtcgctgta acacggcaaa taccgtgcat tgcagcggtt ctggatggcgttcttcgtac gtttgctgcg cccattcttc cctgttccat cgcggtgcgg ccatggcgggcgtctgctct tcacccggca tccaatgaac atgtctctgt cacgcattgt cttggcggcgcccctgcgcc gcaccacact ggccatggcg ctgggcgcgc tgggcgccgc gcccgccgcgtacgccgact ggaacaacca gtccatcatc aaggccggcg agcgccagca cggcatccacatcaagcaaa gcgatggcgc cggcgtacgg accgccaccg gaacgaccat caaggtaagcggtcgtcagg cccagggcgt cctgctggaa aatcccgcgg ccgagctgcg gttccagaacggcagcgtca cgtcttcggg acagctgttc gacgaaggcg tccggcgctt tctgggcaccgtcaccgtca aggccggcaa gctggtcgcc gatcacgcca cgctggccaa cgtcagcgacacccgggacg acgacggcat cgcgctctat gtggccggcg agcaggccca ggccagcatcgccgacagca ccctgcaggg cgcgggcggc gtgcgggtcg agcgcggcgc caatgtcacggtccaacgca gcaccatcgt tgacgggggc ttgcatatcg gcaccctgca gccgctgcagccggaagacc ttccgcccag ccgggtggtg ctgggcgaca ccagcgtgac cgccgtgcccgccagcggcg cgcccgcggc ggtgtctgta ttcggggcca atgagcttac ggttqatggcgggcacatca ccggggggcg ggcagcgggg gtggcggcca tggacggggc gatcgtgcatctg[cagcgcg cgacgatacg gcggggggac gcgcctgccg gcggtgcggt tccaggcggtgctgttcccg gcggcttcgg ccccctcctt gacggctggt atggcgtgga tgtatcggattccaccgtgg acctcgctca g]*tcgatcgtc gaggcgccgc agctgggcgc cgcgatccgggcgggccgcg gcgccagggt gacggtgtcg ggcggcagct tgtccgcacc gcacggcaatgtcatcgaga ccggcggcgg cgcgcgtcgc ttcccgcctc cggcctcgcc cctgtcgatcaccttgcagg cgggcgcacg ggcgcagggg agggcgctgc tgtaccgggt cctgccggagcccgtgaagc tgacgctggc gggcggcgcc caggggcagg gcgacatcgt cgcgacggagctgcctccca ttccaggcgc gtcgagcggg ccgctcgacg tggcgctggc cagccaggcc *Region I cgatggacgg gcgctacccg cgcggtcgac tcgctgtcca tcgacaacgccacctgggtc atgacggaca actcgaacgt cggcgcgctg cggctggcca gcgacggcagcgtcgatttc cagcagccgg ccgaagctgg gcggttcaag tgcctgatgg tcgatacgctggcgggttcg gggctgttcc gcatgaatgt cttcgcggac ctggggctga gcgacaagctggtcgtcatg cgggacgcca gcggccagca caggctgttg gtccgcaaca gcggcagcgagccggccagc ggcaacacca tgctgctggt gcagacgcca cgaggcagcg cggcgacctttacccttgcc aacaaggacg gcaaggtcga tatcggtacc taccgctatc gattggccgccaacggcaat gggcagtgga gcctggtg[gg cgcgaaggcg ccgccggcgc ccaagcccgcgccgcagccc ggtccccagc ccggtcccca gccgccgcag ccgccgcagc cgccgcagccgccacagagg cagccggaag cgccggcgcc gcaaccgccg gcgggcaggg agttgtccgccgcc]**gccaac gcggcggtca acacgggtgg ggtgggcctg gccagcacgc tctggtacgccgaaagcaat gcgttgtcca agcgcctggg cgagttgcgc ctgaatccgg acgccggcggcgcttggggc cgcggcttcg cgcaacgcca gcaactggac aaccgcgccg ggcggcgcttcgaccagaag gtggccggct tcgagctggg cgccgaccac gcggtggcgg tggccggcgggcgctggcac ctgggcgggc tggccggcta tacgcgcggc gaccgcggct ttaccggcgacggcggcggc cacaccgaca gcgtgcatgt cgggggctat gccacctata tcgccaacagcggtttctac ctggacgcga cgctgcgcgc cagccgcctc gaaaatgact tcaaggtggcgggcagcgat gggtacgcgg tcaagggcaa gtaccgcacc catggggtag gcgcctcgctcgaggcgggc cggcgcttcg cccatgccga cggctggttc ctcgagccgc aggccgagctggcggtgttc cgggtcggcg gcggttcgta ccgcgcggcc aatggcctgc gggtgcgcgacgaaggcggc agctcggtgc tgggtcgcct gggcctggag gtcggcaagc gcatcgaactggcaggcggc aggcaggtgc agccatacat caaggccagc gtgctgcagg agttcgacggcgcgggtacg gtacgcacca acggcatcgc gcaccgcacc gaactgcgcg gcacgcgcgccgaactgggc ctgggcatgg ccgccgcgct gggccgcggc cacagcctgt atgcctcgtacgagtactcc aagggcccga agctggccat gccgtggacc ttccacgcgg gctaccggtacagctggtaa ** Region II agcgagaagg gtccatcccc ccgcggggga gattttcctggaggttggcc ggtgccagtc tccaggctca ggcggccagg gcgtgcgggc cgggcaggccgtgctggtgc tggccgaacc B. bronchiseptica p.68 pertactin protein [SEQ IDNO:4] MNMSLSRIVL AAPLRRTTLA MALGALGAAP AAYADWNNQS IIKAGERQHG IHIKQSDGAGVRTATGTTIK VSGRQAQGVL LENPAAELRF QNGSVTSSGQ LFDEGVRRFL GTVTVKAGKLVADHATLANV SDTRDDDGIA LYVAGEQAQA SIADSTLQGA GGVRVERGAN VTVQRSTIVDGGLHIGTLQP LQPEDLPPSR VVLGDTSVTA VPASGAPAAV SVFGANELTV DGGHITGGRAAGVAAMDGAI VHL[QRATIRR GDAPAGGAVP GGAVPGGFGP LLDGWYGVDV SDSTVDLAQ]*SIVEAPQLGAA IRAGRGARVT VSGGSLSAPH GNVIETGGGA RRFPPPASPL SITLQAGARAQGRALLYRVL PEPVKLTLAG GAQGQGDIVA TELPPIPGAS SGPLDVALAS QARWTGATRAVDSLSIDNAT WVMTDNSNVG ALRLASDGSV DFQQPAEAGR FKCLMVDTLA GSGLFRMNVFADLGLSDKLV VMRDASGQHR LLVRNSGSEP ASGNTMLLVQ TPRGSAATFT LANKDGKVDIGTYRYRLAAN GNGQWSLV[GA KAPPAPKPAP QPGPQPGPQP PQPPQPPQPP QRQPEAPAPQPPAGRELSAA]** ANAAVNTGGV GLASTLWYAE SNALSKRLGE LRLNPDAGGA WGRGFAQRQQLDNRAGRRFD QKVAGFELGA DHAVAVAGGR WHLGGLAGYT RGDRGFTGDG GGHTDSVHVGGYATYIANSG FYLDATLRAS RLENDFKVAG SDGYAVKGKY RTHGVGASLE AGRRFAHADGWFLEPQAELA VFRVGGGSYR AANGLRVRDE GGSSVLGRLG LEVGKRIELA GGRQVQPYIKASVLQEFDGA GTVRTNGIAH RTELRGTRAE LGLGMAAALG RGHSLYASYE YSKGPKLAMPWTFHAGYRYS W *  Region I ** Region II B. pertussis p.69 gene [SEQ IDNO:2] atgaacatgt ctctgtcacg cattgtcaag gcggcgcccc tgcgccgcac cacgctggccatggcgctgg gcgcgctggg cgccgccccg gcggcgcatg ccgactggaa caaccagtccatcgtcaaga ccggtgagcg ccagcatggc atccatatcc agggctccga cccgggcggcgtacggaccg ccagcggaac caccatcaag gtaagcggcc gtcaggccca gggcatcctgctagaaaatc ccgcggccga gctgcagttc cggaacggca gtgtcacgtc gtcgggacagttgtccgacg atggcatccg gcgctttctg ggcaccgtca ccgtcaaggc cggcaagctggtcgccgatc acgccacgct ggccaacgtt ggcgacacct gggacgacga cggcatcgcgctctatgtgg ccggcgaaca ggcccaggcc agcatcgccg acagcaccct gcagggcgctggcggcgtgc agatcgagcg cggcgccaat gtcacggtcc aacgcagcgc catcgtcgacgggggcttgc atatcggcgc cctgcagtca ttgcagccgg aagaccttcc gcccagccgggtggtgctgc gcgacaccaa cgtgaccgcc gtgcccgcca gcggcgcgcc cgcggcggtgtctgtgttgg gggccagtga gcttacgctc gacggcgggc acatcaccgg cgggcgggcagcgggggtgg cggccatgca aggggcggtc gtgcatctg[c agcgcgcgac gatacggcgcggggacgcgc ctgccggcgg tgcggttccc ggcggtgcgg ttcccggtgg tgcggttcccggcggcttcg gtcccggcgg cttcggtccc gtcctcgacg gctggtatgg cgtggacgtatcgggctcca gcgtggagct cgcccag]*tcg atcgtcgagg cgccggagct gggcgccgcaatccgggtgg gccgcggcgc cagggtgacg gtgtcgggcg gcagcttgtc cgcaccgcacggcaatgtca tcgagaccgg cggcgcgcgt cgctttgcgc ctcaagccgc gcccctgtcgatcaccttgc aggccggcgc gcatgcccag gggaaagcgc tgctgtaccg ggtcctgccggagcccgtga agctgacgct gaccgggggc gccgatgcgc agggcgacat cgtcgcgacggagctgccct ccattcccgg cacgtcgatc gggccgctcg acgtggcgct ggccagccaggcccgatgga cgggcgctac ccgcgcggtc gactcgctgt ccatcgacaa cgccacctgggtcatgacgg acaactcgaa cgtcggtgcg ctacggctgg ccagcgacgg cagcgtcgat *Region I ttccagcagc cggccgaagc tgggcggttc aaggtcctga cggtcaatacgctggcgggt tcggggctgt tccgcatgaa tgtcttcgcg gacctggggc tgagcgacaagctggtcgtc atgcaggacg ccagcggcca gcacaggctg tgggtccgca acagcggcagcgagccggcc agcgccaaca ccctgctgct ggtgcagacg ccacgaggca gcgcggcgacctttaccctt gccaacaagg acggcaaggt cgatatcggt acctatcgct atcgattggccgccaacggc aatgggcagt ggagcctggt g[ggcgcgaag gcgccgccgg cgcccaagcccgcgccgcag ccgggtcccc agccgccgca gccgccgcag ccgcagccgg aagcgccggcgccgcaaccg ccggcgggca gggagttgtc cgccgcc]**gcc aacgcggcgg tcaacacgggtggggtgggc ctggccagca cgctctggta cgccgaaagc aatgcgttgt ccaagcgcctgggcgagttg cgcctgaatc cggacgccgg cggcgcctgg ggccgcggct tcgcgcaacgccagcagctg gacaaccgcg ccgggcggcg cttcgaccag aaggtggccg gcttcgagctgggcgccgac cacgcggtgg cggtggccgg cggacgctgg cacctgggcg ggctggccggctatacgcgc ggcgaccgcg gcttcaccgg cgacggcggc ggccacaccg acagcgtgcatgtcgggggc tatgccacat atatcgccga cagcggtttc tacctggacg cgacgctgcgcgccagccgc ctggagaatg acttcaaggt ggcgggcagc gacgggtacg cggtcaagggcaagtaccgc acccatgggg tgggcgcctc gctcgaggcg ggccggcgct ttacccatgccgacggctgg ttcctcgagc cgcaggccga gctggcggta ttccgggccg gcggcggtgcgtaccgcgcg gccaacggcc tgcgggtgcg cgacgaaggc ggcagctcgg tgctgggtcgcctgggcctg gaggtcggca agcgcatcga actggcaggc ggcaggcagg tgcagccatacatcaaggcc agcgtgctgc aggagttcga cggcgcgggt acggtacaca ccaacggcatcgcgcaccgc accgaactgc gcggcacgcg cgccgaactg ggcctgggca tggccgccgcgctgggccgc ggccacagcc tgtatgcctc gtacgagtac tccaagggcc cgaagctggccatgccgtgg accttccacg cgggctaccg gtacagctgg taa ** Region II B.pertussis p.69 protein [SEQ ID NO:5] MNMSLSRIVK AAPLRRTTLA MALGALGAAPAAHADWNNQS IVKTGERQHG IRIQGSDPGG VRTASGTTIK VSGRQAQGIL LENPAAELQFRNGSVTSSGQ LSDDGIRRFL GTVTVKAGKL VADHATLANV GDTWDDDGIA LYVAGEQAQASIADSTLQGA GGVQIERGAN VTVQRSAIVD GGLHIGALQS LQPEDLPPSR VVLRDTNVTAVPASGAPAAV SVLGASELTL DGGHITGGRA AGVAAMQGAV VHL[QRATIRR GDAPAGGAVPGGAVPGGAVP GGFGPGGFGP VLDGWYGVDV SGSSVELAQ]*S IVEAPELGAA IRVGRGARVTVSGGSLSAPH GNVIETGGAR RFAPQAAPLS ITLQAGAHAQ GKALLYRVLP EPVKLTLTGGADAQGDIVAT ELPSIPGTSI GPLDVALASQ ARWTGATRAV DSLSIDNATW VMTDNSNVGALRLASDGSVD FQQPAEAGRF KVLTVNTLAG SGLFRMNVFA DLGLSDKLVV MQDASGQHRLWVRNSGSEPA SANTLLLVQT PRGSAATFTL ANKDGKVDIG TYRYRLAANG NGQWSLV[GAKAPPAPKPAPQ PGPQPPQPPQ PQPEAPAPQP PAGRELSAA]**A NAAVNTGGVG LASTLWYAESNALSKRLGEL RLNPDAGGAW GRGFAQRQQL DNRAGRRFDQ KVAGFELGAD HAVAVAGGRWHLGGLAGYTR GDRGFTGDGG GHTDSVHVGG YATYIADSGF YLDATLRASR LENDFKVAGSDGYAVKGKYR THGVGASLEA GRRFTHADGW FLEPQAELAV FRAGGGAYRA ANGLRVRDEGGSSVLGRLGL EVGKRIELAG GRQVQPYIKA SVLQEFDGAG TVHTNGIAHR TELRGTRAELGLGMAAALGR GHSLYASYEY SKGPKLAMPW TFHAGYRYSW *  Region I ** Region II B.parapertussis p.70 gene [SEQ ID NO:3] atcgatgatg cgtcgctgta acacggcaaataccgtgcat tgcagcggtt ctggatggcg ttcttcgtac gtttgctgcg cccattcttccctgttccat cgcggtgcgg gcatggcggg cgtctgctct tcacccggca tccaatgaacatgtctctgt cacgcattgt caaggcggcg cccctgcgcc gcaccacact ggccatggcgctgggcgcgc tgggcgccgc gcccgccgcg tacgccgact ggaacaacca gtccatcatcaaggccggcg agcgccagca cggcatccac atcaagcaaa gcgatggcgc cggcgtacggaccgccaccg gaacgaccat caaggtaagc ggtcgtcagg cccagggcgt cctgctggaaaatcccgcgg ccgagctgcg gttccagaac ggcagcgtca cgtcttcggg acagctgttcgacgaaggcg tccggcgctt tctgggcacc gtcaccgtca aggccggcaa gctggtcgccgatcacgcca cgctggccaa cgtcagcgac acccgggacg acgacggcat cgcgctctatgtggccggcg agcaggccca ggccagcatc gccgacagca ccctgcaggg cgcgggcggcgtgcgggtcg agcgcggcgc caatgtcacg gtccaacgca gcaccatcgt tgacgggggcttgcatatcg gcaccctgca gccgctgcag ccggaagacc ttccgcccag ccgggtggtgctgggcgaca ccagcgtgac cgccgtgccc gccagcggcg cgcccgcggc ggtgtttgtattcggggcca atgagcttac ggttgatggc gggcacatca ccggggggcg ggcagcgggggtggcggcca tggacggggc gatcgtgcat ctg[cagcgcg cgacgatacg gcggggggacgcgcctgccg gcggtgcggt tccaggcggt gcggttcccg gcggtgccgt tcccggcggcttcggccccc tccttgacgg ctggtatggc gtggatgtat cggactccac cgtggacctcgctcag]*tcga tcgtcgaggc gccgcagctg ggcgccgcga tccgggcggg ccgcggcgccagggtgacgg tgtcgggcgg cagcttgtcc gcaccgcacg gcaatgtcat cgagaccggcggcggtgcgc gtcgcttccc gcctccggcc tcgcccctgt cgatcacctt gcaggcgggcgcacgggcgc aggggagggc gctgctgtac cgggtcctgc cggagcccgt gaagctgacgctggcgggcg gcgcccaggg gcagggcgac atcgtcgcga cggagctgcc tcccattocaggcgcgtcga gcgggccgct cgacgtggcg ctggccagcc aggcccgatg gacgggcgctacccgcgcgg tcgactcgct gtccatcgac aacgccacct gggtcatgac ggacaactcgaacgtcggcg cgctgcggct ggccagcgac ggcagcgtcg atttccagca gccggccgaagctgggcggt tcaaggtcct gatggtcgat acgctggcgg gttcggggct gttccgcatgaatgtcttcg cggacctggg gctgagcgac aagctggtcg tcatgcggga cgccagcggccagcacaggc tgtgggtccg caacagcggc agcgagccgg ccagcggcaa caccatgctgctggtgcaga cgccacgagg cagcgcggcg * Region I acctttaccc ttgccaacaaggacggcaag gtcgatatcg gtacctaccg ctatcgattg gccgccaacg gcaatgggcagtggagcctg gtg[ggcgcga aggcgccgcc ggcgcccaag cccgcgccgc agcccggtccccagcccggt ccccagccgc cgcagccgcc gcagccgccg cagccgccgc agccgccgcagccgccacag aggcagccgg aagcgccggc gccgcaaccg ccggcgggca gggagttgtccgccgcc]**gcc aacgcggcgg tcaacacggg tggggtgggc ctggccagca cgctctggtacgccgaaagc aatgcgttgt ccaagcgcct gggcgagttg cgcctgaatc cggacgccggcggcgcttgg ggccgcggct tcgcgcaacg ccagcaactg gacaaccgcg ccgggcggcgcttcgaccag aaggtggccg gcttcgagct gggcgccgac cacgcggtgg cggtggccggcgggcgctgg cacctgggcg ggctggccgg ctatacgcgc ggcgaccgcg gctttaccggcgacggcggc ggccacaccg acagcgtgca tgtcgggggc tatgccacct atatcgccaacagcggtttc tacctggacg cgacgctgcg cgccagccgc ctcgaaaatg acttcaaggtggcgggcagc gatgggtacg cggtcaaggg caagtaccgc acccatgggg taggcgtctcgctcgaggcg ggccggcgct tcgcccatgc cgacggctgg ttcctcgagc cgcaggccgagctggcggtg ttccgggtcg gcggcggtgc gtaccgcgcg gccaatggcc tgcgggtgcgcgacgaaggc ggcagctcgg tgctgggtcg cctgggcctg gaggtcggca agcgcatcgaactggcaggc ggcaggcagg tgcagccata catcaaggcc agcgtgttgc aggagttcgacggcgcgggt acggtacgca ccaacggcat cgcgcatcgc accgaactgc gcggcacgcgcgccgaactg ggcctgggca tggccgccgc gctgggccgc ggccacagcc tgtatgcctcgtacgagtac tccaagggcc cgaagctggc catgccgtgg accttccacg cgggctaccggtacagctgg taaagcgaga agggtccatc ccccgcggag gagtttttcc tggaggttggccggtgccag tctccaggct caggcggcca gggcctgcgg gccgggcagg ccgtgctggtgctggccgaa ccattgcaca gggtgttcgg ccaagggcgg cgacttcgcc gatgaccagcaacgccgggg ggcgcacgct gcgccggcgc gcgatc ** Region I B. parapertussisp.70 protein [SEQ ID NO:6] MNMSLSRIVK AAPLRRTTLA MALGALGAAP AAYADWNNQSIIKAGERQHG IHTKQSDGAG VRTATGTTIK VSGRQAQGVL LENPAAELRF QNGSVTSSGQLFDEGVRRFL GTVTVKAGKL VADHATLANV SDTRDDDGIA LYVAGEQAQA SIADSTLQGAGGVRVERGAN VTVQRSTIVD GGLHIGTLQP LQPEDLPPSR VVLGDTSVTA VPASGAPAAVFVFGANELTV DGGHITGGRA AGVAAMDGAI VHL[QRATIRR GDAPAGGAVP GGAVPGGAVPGGFGPLLDGW YGVDVSDSTV DLAQ]*SIVEAP QLGAAIRAGR GARVTVSGGS LSAPHGNVIETGGGARRFPP PASPLSITLQ AGARAQGRAL LYRVLPEPVK LTLAGGAQGQ GDIVATELPPIPGASSGPLD VALASQARWT GATRAVDSLS IDNATWVMTD NSNVGALRLA SDGSVDFQQPAEAGRFKVLM VDTLAGSGLF RMNVFADLGL SDKLVVMRDA SGQHRLWVRN SGSEPASGNTMLLVQTPRGS AATFTLANKD GKVDIGTYRY RLAANGNGQW SLV[GAKAPPA PKPAPQPGPQPGPQPPQPPQ PPQPPQPPQP PQRQPEAPAP QPPAGRELSA A]**ANAAVNTGG VGLASTLWYAESNALSKRLG ELRLNPDAGG AWGRGFAQRQ QLDNRAGRRF DQKVAGFELG ADHAVAVAGGRWHLGGLAGY TRGDRGFTGD GGGHTDSVHV GGYATYIANS GFYLDATLRA SRLENDFKVAGSDGYAVKGK YRTHGVGVSL EAGRRFAHAD GWFLEPQAEL AVFRVGGGAY RAANGLRVRDEGGSSVLGRL GLEVGKRIEL AGGRQVQPYI KASVLQEFDG AGTVRTNGIA HRTELRGTRAELGLGMAAAL GRGHSLYASY EYSKGPKLAM PWTFHAGYRY SW *  Region I ** Region II

What is claimed is:
 1. An immunogenic composition comprising a mixtureof pertactins of Bordetella species, wherein said composition comprises:(a) pertactin of Bordetella parapertussis, and (b) pertactin ofBordetella bronchiseptica, in amounts sufficient to induce a humoral orcellular immune response against Bordetella parapertussis and Bordetellabronchiseptica in an animal to which the immunogenic composition isadministered.
 2. An immunogenic composition as claimed in claim 1, whichalso comprises pertactin of Bordetella pertussis in an amount sufficientto induce a humoral or cellular immune response against Bordetellapertussis in an animal to which the immunogenic composition isadministered.
 3. An immunogenic composition comprising a mixture ofpertactins of Bordetella species or fragments thereof, wherein saidpertactins or fragments thereof comprise at least two Bordetellabronchiseptica pertactin variants and wherein said pertactins ofBordetella species or fragments thereof are present in an amountsufficient to induce a humoral or immune response against Bordetellabronchiseptica in an animal to which the composition is administered. 4.An immunogenic composition according to claim 3, comprising a mixture ofpertactins of Bordetella species or fragments thereof, wherein saidpertactins or fragments thereof comprise at least two Bordetellabronchiseptica pertactin variants, and said at least two Bordetellabronchiseptica pertactin variants differ from each other at least intheir Region II.
 5. An immunogenic composition comprising a mixture ofpertactins of Bordetella species or fragments thereof, wherein saidpertactins or fragments thereof comprise a mixture of Bordetellabronchiseptica pertactin variants, wherein each Bordetellabronchiseptica pertactin variant comprises 6, 7, 8, or 9 repeating PQPamino acid sequences in Region II thereof, and wherein said Bordetellabronchiseptica pertactin variants are present in amounts sufficient toinduce a humoral or cellular immune response against Bordetellabronchiseptica in an animal to which the immunogenic composition isadministered.
 6. An immunogenic composition according to claim 5,wherein at least two of the Bordetella bronchiseptica pertactin variantsdiffer from each other at least in the number of repeating PQP aminoacid sequences in their Region II.
 7. An immunogenic composition asclaimed in claim 5, which also comprises pertactins of Bordetellaparapertussis or fragment thereof, Bordetella pertussis or fragmentthereof, or mixtures thereof, in amounts sufficient to induce a humoralor cellular immune response against Bordetella parapertussis orBordetella pertussis in an animal to which the immunogenic compositionis administered.
 8. An immunogenic composition according to claim 3,comprising a mixture of pertactins of Bordetella species or fragmentsthereof, wherein said pertactins or fragments thereof comprise at leasttwo Bordetella bronchiseptica pertactin variants, and said at least twoBordetella bronchiseptica pertactin variants differ from each other atleast in their Region I.
 9. An immunogenic composition comprising amixture of pertactins of Bordetella species or fragments thereof,wherein said pertactins or fragments thereof comprise a mixture ofBordetella bronchiseptica pertactin variants, wherein each Bordetellabronchiseptica pertactin variant comprises 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof, and wherein said Bordetellabronchiseptica pertactin variants are present in amounts sufficient toinduce a humoral or cellular immune response against Bordetellabronchiseptica in an animal to which the immunogenic composition isadministered.
 10. An immunogenic composition according to claim 9,wherein at least two of the Bordetella bronchiseptica pertactin variantsdiffer at least from the number of repeating GGXXP amino acid sequencesin their Region I.
 11. An immunogenic composition as claimed in claim 9,which also comprises pertactins of Bordetella parapertussis or afragment thereof, Bordetella pertussis or a fragment thereof, ormixtures thereof, in amounts sufficient to induce a humoral or cellularimmune response against Bordetella parapertussis or Bordetella pertussisin an animal to which the immunogenic composition is administered. 12.An immunogenic composition comprising a mixture of pertactins ofBordetella bronchiseptica species or fragments thereof, wherein saidpertactins or fragments thereof comprise a mixture of Bordetellabronchiseptica pertactin variants, wherein at least one of saidBordetella bronchiseptica pertactin variants comprises Region II ofpertactin of Bordetella bronchiseptica having 6, 7, 8, or 9 repeatingPQP amino acid sequences in Region II thereof, and at least another ofsaid Bordetella bronchiseptica pertactin variants comprises Region I ofpertactin of Bordetella bronchiseptica having 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof.
 13. An immunogenic compositioncomprising a mixture of fragments of pertactins of Bordetella species,wherein said composition comprises: (a) pertactin of Bordetellaparapertussis or a fragment thereof, and (b) pertactin of Bordetellabronchiseptica or a fragment thereof containing Region I, Region II, orboth Region I and Region II, in amounts sufficient to induce a humoralor cellular immune response against Bordetella parapertussis andBordetella bronchiseptica in an animal to which the immunogeniccomposition is administered.
 14. An immunogenic composition as claimedin claim 13, which also comprises pertactin of Bordetella pertussis or afragment thereof in an amount sufficient to induce a humoral or cellularimmune response against Bordetella pertussis in an animal to which thecomposition is administered.
 15. A composition comprising a mixture ofat least two Bordetella bronchiseptica pertactin variants, wherein eachvariant comprises Region II of a pertactin of Bordetella bronchiseptica,and wherein said variants differ from each other at least in the RegionII they each comprise.
 16. A composition comprising a mixture ofBordetella bronchiseptica pertactin variants, wherein each Bordetellabronchiseptica pertactin variant comprises 6, 7, 8, or 9 repeating PQPamino acid sequences in Region II thereof, and at least two Bordetellabronchiseptica pertactin variants differ in the number of said repeatingPQP amino acid sequences contained therein.
 17. A composition as claimedin claim 16, which also comprises pertactins of Bordetella parapertussisor a fragment thereof, Bordetella pertussis or a fragment thereof, ormixtures thereof.
 18. A composition comprising a mixture of at least twoBordetella bronchiseptica pertactin variants, wherein each Bordetellabronchiseptica pertactin variant comprises Region I of pertactin ofBordetella bronchiseptica, and wherein said variants differ from eachother at least in the Region I that each comprises.
 19. A compositioncomprising a mixture of Bordetella bronchiseptica pertactin variants,wherein each Bordetella bronchiseptica pertactin variant comprises 1, 2,or 3 repeating GGXXP amino acid sequences in Region I thereof, and atleast two Bordetella bronchiseptica pertactin variants differ in thenumber of said repeating GGXXP amino acid sequences contained therein.20. A composition as claimed in claim 19, which also comprisespertactins of Bordetella parapertussis or a fragment thereof, Bordetellapertussis or a fragment thereof, or mixtures thereof.
 21. A compositioncomprising a mixture of Bordetella bronchiseptica pertactin variants,wherein one of said Bordetella bronchiseptica pertactin variantscomprises Region II of pertactin of Bordetella bronchiseptica andanother of said Bordetella bronchiseptica pertactin variants comprisesRegion I of pertactin of Bordetella bronchiseptica.
 22. The compositionaccording to claim 21, wherein said Region II has 6, 7, 8, or 9repeating PQP amino acid sequences.
 23. The composition according toclaim 21, wherein said Region I has 1, 2, or 3 repeating GGXXP aminoacid sequences.
 24. A composition comprising a mixture of Bordetellabronchiseptica pertactin variants, wherein one of said Bordetellabronchiseptica pertactin variants comprises Region II of pertactin ofBordetella bronchiseptica having 6, 7, 8, or 9 repeating PQP amino acidsequences in Region II thereof, and another of said Bordetellabronchiseptica pertactin variants comprises Region I of pertactin ofBordetella bronchiseptica having 1, 2, or 3 repeating GGXXP amino acidsequences in Region I thereof.
 25. A composition as claimed in claim 24,which also comprises pertactins of Bordetella parapertussis or afragment thereof, Bordetella pertussis or a fragment thereof, ormixtures thereof.
 26. A polypeptide comprising a sequence or a fragmentof said sequence selected from the group consisting of SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ IDNO:22.
 27. A polypeptide consisting of the amino acids in SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, orSEQ ID NO:22.
 28. A polynucleotide encoding a polypeptide as claimed inclaim
 26. 29. A purified DNA or RNA sequence that hybridizes undermoderate or high stringency conditions to the polynucleotide of claim 28or at least to 15 nucleotides thereof.
 30. A polynucleotide encoding apolypeptide as claimed in claim
 27. 31. A purified DNA or RNA sequencethat hybridizes under moderate or high stringency conditions to thepolynucleotide of claim 30 or at least to 15 nucleotides thereof. 32.Purified antibodies that bind to a polypeptide of claim
 26. 33. Purifiedantibodies according to claim 32, wherein the antibodies are monoclonalantibodies.
 34. Purified antibodies according to claim 32, wherein theantibodies are polyclonal antibodies.
 35. An immunological complexcomprising a polypeptide of claim 26 and an antibody that specificallyrecognizes said polypeptide.
 36. A method for detecting infection byBordetella, wherein the method comprises providing a compositioncomprising a biological material suspected of being infected withBordetella, and assaying for the presence of a polypeptide of claim 26.37. The method as claimed in claim 36, wherein the polypeptide isassayed by electrophoresis or by immunoassay with antibodies that areimmunologically reactive with the polypeptide.
 38. An in vitrodiagnostic method for the detection of the presence or absence ofantibodies, which bind to an antigen comprising a polypeptide of claim26, wherein the method comprises contacting the antigen with abiological fluid for a time and under conditions sufficient for theantigen and antibodies in the biological fluid to form anantigen-antibody complex, and detecting the formation of the complex.39. The method as claimed in claim 38, which further comprises measuringthe formation of the antigen-antibody complex.
 40. The method as claimedin claim 38, wherein the formation of antigen-antibody complex isdetected by immunoassay based on Western blot technique, ELISA, indirectimmunofluorescence assay, or immunoprecipitation assay.
 41. A diagnostickit for the detection of the presence or absence of antibodies, whichbind a polypeptide of claim 26 or mixtures thereof, wherein the kitcomprises an antigen comprising polypeptide of claim 26 or mixtures ofsaid polypeptides, and means for detecting the formation of immunecomplex between the antigen and antibodies, wherein the means arepresent in an amount sufficient to perform said detection.
 42. Animmunogenic composition comprising at least one polypeptide of claim 26in an amount sufficient to induce an immunogenic or protective responsein vivo, and a pharmaceutically acceptable carrier therefor.
 43. Theimmunogenic composition as claimed in claims 1 to 14, wherein saidcomposition comprises a neutralizing amount of at least one polypeptideof claim
 26. 44. An immunogenic composition comprising a polynucleotideaccording to any one of claims 28 to
 31. 45. A vaccine comprising animmunogenic composition according to any one of claims 1 to 14, 42, 43or 44 along with a pharmaceutically acceptable vehicle.
 46. A method fordetecting the presence or absence of Bordetella comprising: (1)contacting a sample suspected of containing genetic material ofBordetella with at least one nucleotide probe, and (2) detectinghybridization between the nucleotide probe and the genetic material inthe sample, wherein said nucleotide probe is complementary to apolynucleotide sequence as claimed in any one of claim 28 or
 30. 47. Avaccination kit comprising at least an immunogenic composition accordingto claims 1 to 14 or 42 to 44, and means for administering thecomposition to an animal.
 48. An immunogenic composition consistingessentially of: (A) a polypeptide comprising Region I and Region II, orone polypeptide comprising Region I and one polypeptide comprisingRegion II, of a pertactin of Bordetella pertussis; (B) a polypeptidecomprising Region I and Region II, or one polypeptide comprising RegionI and one polypeptide comprising Region II, of a pertactin of Bordetellaparapertussis; (C) a polypeptide comprising Region I and Region II, orone polypeptide comprising Region I and one polypeptide comprisingRegion II, of a pertactin of Bordetella bronchiseptica strain 9.73 and apolypeptide comprising Region I and Region II, or one polypeptidecomprising Region I and one polypeptide comprising Region II, of apertactin of Bordetella bronchiseptica of strain SEI.
 49. An immunogeniccomposition consisting essentially of: (A) a pertactin of Bordetellabronchiseptica; (B) FHA of Bordetella bronchiseptica; and (C) apertactin of Bordetella parapertussis.
 50. The immunogenic compositionas claimed in claim 49, wherein the pertactin of Bordetellabronchiseptica is from strain 9.73.
 51. The immunogenic composition asclaimed in claim 49, wherein the FHA of Bordetella bronchiseptica isfrom strain 9.73.
 52. An immunogenic composition as claimed in any oneof claims 1 to 14, 42 to 44, or 48 to 51, wherein the compositionfurther comprises at least one adhesin of Bordetella selected from thegroup consisting of FHA, AGG2, AGG3, and/or at least one toxin ofBordetella selected from the group consisting of PTX, DNT, TCT, Ac-Hly.53. A DNA chip, wherein said chip comprises at least one polynucleotideaccording to claims 28 to 31 or fragment thereof.
 54. Use of monoclonalantibodies according to claim 33 for treating Bordetella infections. 55.A microarray comprising microbeads, wherein said microbeads each bearsmultiple copies of a polynucleotide according to any one of claims 28 to31 or a fragment thereof, and wherein the polynucleotide or fragmentthereof is different from one microbead to another.